U.S. patent number 4,736,018 [Application Number 06/779,287] was granted by the patent office on 1988-04-05 for blood coagulation inhibiting proteins, processes for preparing them and their uses.
This patent grant is currently assigned to Boehringer Ingelheim International GmbH. Invention is credited to Christian P. M. Reutelingsperger.
United States Patent |
4,736,018 |
Reutelingsperger |
April 5, 1988 |
Blood coagulation inhibiting proteins, processes for preparing them
and their uses
Abstract
This invention discloses proteins which inhibit the coagulation
of the blood, processes for preparing these proteins, and the use
thereof.
Inventors: |
Reutelingsperger; Christian P.
M. (Maastricht, NL) |
Assignee: |
Boehringer Ingelheim International
GmbH (DE)
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Family
ID: |
26645980 |
Appl.
No.: |
06/779,287 |
Filed: |
September 23, 1985 |
Foreign Application Priority Data
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Sep 21, 1984 [NL] |
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8402904 |
Mar 4, 1985 [NL] |
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8500601 |
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Current U.S.
Class: |
530/381;
514/13.7; 530/413; 530/420; 530/412; 530/416; 530/827 |
Current CPC
Class: |
A61K
35/44 (20130101); A61P 7/02 (20180101); Y10S
530/829 (20130101); Y10S 530/827 (20130101) |
Current International
Class: |
A61K
35/44 (20060101); C07K 015/06 (); A61K
037/02 () |
Field of
Search: |
;530/380,381,412,413,416,420,827,829 ;424/101 ;514/2,21 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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2353318 |
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Jan 1973 |
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DE |
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1443189 |
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Oct 1972 |
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GB |
|
Other References
Schapira et al., "Protection of Human Plasma Kallikrein from
Inactivation by C1 Inhibitor and Other Protease Inhibitors",
Biochemistry, 1981, 20, 2738-2743. .
Miller-Andersson et al., "Purification of Antithrombin III by
Affinity Chromatography", Thrombosis Research, vol. 5, pp. 439-452,
1974, Pergamon Press, Inc. .
Travis et al., "Human Plasma Proteinase Inhibitors", Ann. Rev.
Biochem., 1983, 52:655-709. .
Biological Abstracts, 53441. .
Chemical Abstracts, 100:83398T..
|
Primary Examiner: Phillips; Delbert R.
Assistant Examiner: Nutter; Nathan M.
Attorney, Agent or Firm: Saidman, Sterne, Kessler &
Goldstein
Claims
What is claimed as new and intended to be secured by Letters Patent
is:
1. A process for preparing an anti-coagulant protein from tissue
which comprises:
(a) homogenizing said tissue, differentially centrifuging said
homogenized tissue, and subjecting the supernatant liquid to one or
more of the following purification treatments in any desired
sequence:
(b) precipitation with salt,
(c) affinity chromatography,
(d) ion exchange chromatography,
(e) chromatography using a molecular sieve;
wherein said tissue is selected from the group consisting of blood
vessel walls, highly vascularized tissue, or endothelial cell
cultures.
2. The process of claim 1 wherein said anti-coagulant protein is
further purified using immunoabsorption chromatography.
3. The process of claim 1 wherein said anti-coagulant protein is
purified using phospholipid vesicles.
4. The process of claim 1 wherein ammonium sulfate is used for the
precipitation in step (b), hydroxyapatite is used for
chromatography in step (c), DEAE-Sephacel is used for
chromatography in step (d), and Sephadex G-100 or G-75 is used for
chromatography in step (e).
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to proteins which inhibit the coagulation of
the blood, processes for preparing these proteins, and their
use.
2. Description of the Background Art
Anti-coagulant proteins, which are present in most mammals, can be
divided into three groups based on their different mechanisms of
activity.
One group of proteins form a complex with a coagulation factor and
thereby render the coagulation factor inactive. Proteins in this
category include antithrombin III (Thromb. Res., 5: 439-452
(1974)), alpha.sub.1 -protease inhibitor (Ann. Rev. Biochem., 52:
655-709 (1983)), alpha.sub.2 -macroglobulin (Ann. Rev. Biochem.,
52: 655-709 (1983)), C.sub.1 -inhibitor (Biochemistry, 20:
2738-2743 (1981)), and protease nexin (J. Biol. Chem., 258:
10,439-10,444 (1983)).
A second group of proteins act proteolytically on a coagulating
factor and thereby inactivate it. The only protein of this kind
that has been described is protein C (J. Biol. Chem., 251: 355-363
(1976)).
The third category to which anti-coagulant proteins can be grouped
are those which screen and/or hydrolyze the negatively charged
phospholipids so that the phospholipid-dependent reactions of the
blood coagulation mechanism are inhibited. Thus far, only
phospholipases isolated from various types of snake venom have been
described as having this mode of action (Eur. J. Biochem., 112:
25-32 (1980)).
In recent years, the step-wise coagulation system has been
investigated thoroughly. It is understood to be an intensifying
multi-stage system of different interconnected proteolytic
reactions in which an enzyme converts a zymogen into the active
form (cf. Jackson, C. M. and Nemerson, Y., Ann. Rev. Biochem., 49:
765-811 (1980)). The speed of this reaction is decisively increased
by the presence of phospholipids and other cofactors such as factor
V.sub.a and factor VIII.sub.a. In vivo, the procoagulation
reactions are regulated by a variety of inhibitory mechanisms which
prevent an explosively thrombotic trauma after slight activation of
the coagulation cascade.
The mechanisms by which the anti-coagulation proteins of these
three groups act have been described (Rosenberg, R. D. and
Rosenberg, J. S., J. Clin. Invest., 74: 1-6 (1984)).
In Group 1, serine-protease factor X.sub.a and thrombin are
inactivated as a result of their binding to antithrombin III or to
the antithrombin/heparin complex. Both the prothrombin activation
and also the formation of fibrin can be inhibited in this way. In
addition to antithrombin III, there are also various other
plasmaprotease inhibitors such as alpha.sub.2 -macroglobulin and
antitrypsin, the activity of which is dependent on time.
In Group 2, the discovery of protein C led to another
anti-coagulation mechanism. Once protein C is activated, it acts as
an anti-coagulant by selective proteolysis of the protein cofactors
V.sub.a and VIII.sub.a, by which prothrombinase and the enzyme
which converts factor X are deactivated.
In Group 3, plasmin cleaves monomeric fibrin 1, a product of the
effect of thrombin on fibrinogen, thereby preventing the formation
of an insoluble fibrin (Nossel, H. L., Nature, 291: 165-167
(1981)).
Of the above-mentioned native proteins involved in the coagulation
process, at present only antithrombin III is clinically used.
However, the increase in the tendency to bleed when this protein is
administered has proven to be a serious disadvantage.
All the agents previously used as anticoagulants, whether native to
the body or synthetic, in some way render the coagulation factors
ineffective and thereby lead to side effects which have a
disadvantageous effect on the coagulation process.
SUMMARY OF THE INVENTION
It has been found possible to isolate native proteins which have
blood coagulation-inhibiting properties, but do not increase the
risk of bleeding. These proteins lose their inhibiting properties
in the event of major bleeding, so that the normal coagulation
processes can proceed without disruption and there is no danger of
bleeding to death.
The present invention thus relates to anti-coagulant proteins,
hereinafter referred to as VAC (Vascular Anti-Coagulant), which do
not inactivate the coagulation factors. These proteins are capable
of inhibiting the coagulation induced by a vascular procoagulant or
by the factor X.sub.a, but do not inhibit the coagulation induced
by thrombin. In addition, they do not inhibit the biological and
amidolytic activity of factors X.sub.a and II.sub.a.
DESCRIPTION OF THE FIGURES
FIG. 1: Gel Filtration of VAC on Sephadex G-100
The column (3.times.80 cm) was prepared at 60 cm pressure and
equilibrated with 500 mM NaCl and 20 mM Tris/HCl, pH 7.5. The
VAC-containing fraction obtained after DEAE chromatography was
concentrated (2 ml) and then passed over the Sephadex G-100. The
pressure was maintained at 60 cm. and the void volume was 245 ml
(fraction 70). The fractions (2 ml) were dialyzed against
Tris-buffered saline (TBS) containing 10% glycerol, and tested for
VAC activity by the one-stage coagulation test as described in
Example 1. The coagulation times were determined using 1:10
dilutions of the G-100 fractions in TBS. The coagulation time in
the absence of VAC was 65 seconds.
FIG. 2: Analytical SDS-PAGE of VAC
SDS-PAGE gels contained by weight 10% acrylamide, 0.27% of
N,N.sup.3 -methylene-bisacrylamide, and 0.1% SDS (Laemli, U.K.,
Nature, 227: 680-685 (1970)).
Lane 1: reduced reference proteins;
Lane 2: 25 ug reduced VAC;
Lane 3: 25 ug non-reduced VAC.
The gel was stained with Coomassie Blue and decolorized in the
manner described in Example 1.
FIG. 3: Isoelectric pH of VAC
Electrofocusing was carried out with PAG plates in a pH range of
from 3.5-9.5 (see Example 1). 200 ug of human H.sub.b.sup.1 and 20
ug of VAC were applied to the gel after the pH gradient had formed
in the gel. Human H.sub.b was used as a reference (isoelectric
point: pH 6.8). The gel was fixed for 30 minutes with 0.7M
trichloroacetic acid and stained with Coomassie Blue.
FIG. 4: Analysis of the Binding of VAC to Negatively Charged
Phospholipid Liposomes with SDS-PAGE
SDS-PAGE was carried out according to Laemli (Laemli, U.K. Nature,
227: 680-685 (1970)) on the same plates as described in Example 1.
The samples analyzed were obtained from the binding experiments as
mentioned in the explanation to Table B.
Lane 1: reduced reference proteins; Lane 2: supernatant of VAC
preparation centrifuged in the absence of liposomes; Lane 3:
supernatant of VAC preparation centrifuged in the presence of
liposomes; Lane 4: supernatant of VAC preparation centrifuged in
the presence of liposomes and Ca.sup.++ ; Lane 5: supernatant from
liposome precipitate of Lane 4 resuspended in TBS (10 mM EDTA) and
centrifuged.
FIG. 5: Effect of VAC Concentration on Inhibition (%) of Thrombin
Formation
The concentrations of VAC mentioned are the final concentrations
present in the test systems. The thrombin formation was measured in
1 uM prothrombin, 10 nM factor X.sub.a and 0.5M ( ) or 5.0M
(.cndot..cndot.) phospholipid membrane (PC/PS; 4:1, mol/mol) in 10
mM TBSA with CaCl.sub.2. The reaction mixture was stirred with the
specific quantities of VAC (Specific activity: 1300 units/mg) for 3
minutes at 37.degree. C. without prothrombin. By adding prothrombin
to the mixture, as in Example 1, the thrombin formation was
initiated and the speed measured. The speed of thrombin formation
in the absence of VAC was 3.3 nM II.sub.a /min. ( ) or 10.9 nM
II.sub.a /min. (.cndot. .cndot.).
FIG. 6: Effect of Phospholipid Concentration on Inhibition (%) of
Thrombin Formation by VAC
Thrombin formation was measured at 1 um prothrombin, 10 nM factor
X.sub.a, 10.7 ug/ml VAC (Specific activity: 1300 units/mg) and at
various concentrations of phospholipid membrane (PC/PS; 4:1,
mol/mol) in TBSA. Factor X.sub.a, VAC and phospholipid were stirred
in TBSA for 3 minutes at 37.degree. C. The thrombin formation was
initiated by adding prothrombin to the reaction mixture. The rate
of thrombin formation was measured as described in Example 1. The
percent inhibition of thrombin formation (.cndot. .cndot.) was
measured for each phospholipid concentration with the corresponding
rate of thrombin formation in the absence of VAC ( ).
FIG. 7: Gel Filtration of the 10,000.times.g Supernatant of an
Umbilical Cord Artery Homogenate on Sephadex G-100
2 ml of the 10,000.times.g supernatant of a homogenized umbilical
cord was loaded on a Sephadex G-100 column (1.5.times.80 cm), which
was pre-equilibrated with TBS. The column was eluted with TBS.
Aliquots of the resulting fractions were tested in the MPTT.
Certain fractions ( ) express a procoagulant activity and initiated
coagulation in the MPTT without the addition of HTP, factor
X.sub.a, or thrombin. Other distinct fractions ( ) prolong clotting
time in the MPTT, using HTP to initiate coagulation. These
fractions were pooled and further fractionated.
FIG. 8: Chromtography of the Anti-Coagulant on DEAE-Sephacel (A)
and Sephadex G-75 (B).
The pool, containing the anti-coagulant, from the Sephadex G-100
column was applied to DEAE-Sephacel. Elution was performed with a
200 ml linear gradient of 50-300 mM NaCl (- - -). Fractions (4 ml)
were collected. A.sub.280 was measured for each fraction (--) and
anti-coagulant activity assayed in the MPTT using HTP (final
concentration: 95 ug protein/ml) as initiator of coagulation
(.cndot.). The fractions with anti-coagulant activity were pooled,
concentrated, and subsequently applied to Sephadex G-75 (B).
Fractions (2 ml) were collected. Each fraction was measured for
A.sub.280 (--) and anti-coagulant activity (.cndot.). V.sub.o
represents the void volume of the column.
FIG. 9: Dose Response of the Anti-Coagulant in the MPTT
Varying amounts of the anti-coagulant were added to the MPTT.
Coagulation was initiated with HTP (final concentration: 95 ug
protein/ml). Control clotting time was 65 s.
FIG. 10: Gel Electrophoresis of Several Fractions of the G-75
Eluant
Several fractions of the G-75 eluant were analyzed by SDS-PAGE. The
gels were silver-stained according to Merril et al.,
Electrophoresis J., 3: 17-23 (1982)). Lane 1: reduced low molecular
weight standards; Lanes 2-6: unreduced aliquots of the G-75
fractions numbers 35, 39, 41, 43 and 50, respectively.
FIG. 11: The Effect of Proteolytic Enzymes on the Activity of the
Anti-Coagulant
The anti-coagulant was incubated at 37.degree. C. with protease
type I ( , final concentration: 0.11 units/ml), trypsin ( , final
concentration: 88 BAEE units/ml) and without proteolytic enzymes
(.cndot.). At the times indicated, 5 ul containing 6 ug protein of
the anti-coagulant was removed from the reaction mixture and added
to the MPTT. Clotting was initiated with HTP (final concentration:
18 ug protein/ml). Control clotting time was 110 s. The units given
in this legend for the proteolytic enzymes are calculated from the
values supplied by the manufacturer.
FIG. 12: Effect of Vascular Anti-Coagulant on the Clotting Times,
Induced in the MPTT by Either HTP, Factor X.sub.a, or thrombin
The concentrations of the coagulation initiators (HTP: 18 ug
protein/ml, 1.5 nM factor X.sub.a, or 0.4 nM thrombin) were chosen
to give control clotting times of about 110 seconds (open bars).
When factor X.sub.a was used, phospholipid vesicles (final
concentration 10 uM), composed of the Ole.sub.2 Gro-P-Ser/Ole.sub.2
Gro-P-Cho (molar ratio, 20:80) were added to the reaction mixture.
Clotting times induced by the indicated agents in the presence of
3.5 ug anti-coagulant protein are shown by the shaded bars.
FIG. 13: Effect of the Anti-Coagulant of Prothrombin Activation by
(X.sub.a, V.sub.a, phospholipid, CA.sup.2+), (X.sub.a,
phospholipid, Ca.sup.2+), (X.sub.a, Ca.sup.2+).
The reaction mixtures contained: (A) 1 uM prothrombin, 0.3 nM
X.sub.a, 0.6 nM V.sub.a, 0.5 uM phospholipid and 10 mM CaCl.sub.2
with 12.0 ug/ml anti-coagulant ( ), 4.8 ug/ml anticoagulant ( ),
and 0.0 anticoagulant (.cndot.); (B) 1 uM prothrombin, 10 nM
X.sub.a, 0.5 uM phospholipid, and 10 mM CaCl.sub.2 with 2.4 ug/ml
anti-coagulant ( ), 0.48 ug/ml anti-coagulant ( ), and 0.0
anti-coagulant (.cndot.); (C) 1 uM prothrombin, 75 nM X.sub.a, and
10 mM CaCl.sub.2 with 120 ug/ml anti-coagulant ( ), and 0.0
anti-coagulant (.cndot.). At times indicated, samples were removed
and thrombin was determined.
FIG. 14: Immunoblots
Immunoblots were obtained by the procedure described in Example 5.
Lane 1: bovine aorta protein fraction with VAC-activity; Lane 2:
bovine aorta protein fraction with VAC-activity; Lane 3: bovine
lung protein fraction with VAC-activity; Lane 4: human umbilical
cord artery protein fraction with VAC-activity; Lane 5: rat aorta
protein fraction with VAC-activity; and Lane 6: horse aorta protein
fraction with VAC-activity.
FIG. 15: Gel Electrophoresis (A) and Anti-Coagulant Activity (B) of
the Various Fractions of the G-75 Eluate
Various fractions of the G-75 eluate were subjected to gel
electrophoresis as described. The bands were stained with silver by
the method of Merril et al., Electrophoresis J., 3: 17-23 (1982).
Electrophoresis lane 1: low molecular weight standards;
electrophoresis lanes 2-6: equal volumes of non-reduced G-75
fractions with increasing elution volume. Specific quantities of
the G-75 fractions, which had been analyzed by gel electrophoresis,
were tested in the MPTT using HTP to initiate coagulation. The
control coagulation time is represented by the open bar. The
figures under the shaded bars correspond to the numbers of the
electrophoresis lanes in FIG. 15A.
FIG. 16: Heat Inactivation of the Vascular Anti-Coagulation Agent
(VAC)
The anti-coagulation agent was incubated at 56.degree. C. and,
after the various incubation periods, 5 ul samples containing 3.6
ug protein were taken, immediately cooled with ice, and tested in
the MPTT using HPT as coagulation initiator. The coagulation time
of the control sample was 110 seconds.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present invention relates to an agent which has blood
coagulation-inhibiting properties but not the disadvantageous side
effects on the coagulation process which accompany the
anti-coagulants currently known.
The anti-coagulant proteins of the invention do not deactivate the
coagulation factors, but inhibit:
the modified prothrombin-time experiment and/or
the modified activated partial thromoplastin-time experiment
and/or
the non-modified prothrombin-time experiment and/or
the prothrombin activation by the coagulation factor X.sub.a in the
presence of negatively charged phospholipids and Ca.sup.2+
and/or
the intrinsic X-activation by factor IX.sub.a in the presence of
negatively charged phospholipids and Ca.sup.2+ and/or
the prothrombin activation of isolated stimulated blood platelets
and/or
the coagulation induced by the walls of the blood vessels
and/or
the coagulation-dependent platelet aggregation.
The invention also relates to anti-coagulant proteins that do not
inactivate the coagulation factors and whose inhibitory activity
depends on the concentration of phospholipids. The proteins of the
invention induce inhibition of prothrombin activation by factor
X.sub.a. This inhibition depends on the phospholipid concentration
and is less at high phospholipid concentrations. Phospholipids are
not hydrolyzed by the proteins of the invention.
The invention further relates to anti-coagulant proteins which do
not inactivate the coagulation factors and which bind, via the
divalent cations Ca.sup.2+ and/or Mn.sup.2+, to negatively charged
phospholipids, which can be found, for example, in vesicles,
liposomes or etherosomes and/or, via the divalent cations Ca.sup.2+
and/or Mn.sup.2+, to negatively charged phospholipids which are
coupled with Spherocil. The binding of the anticoagulant proteins
of the invention to negatively charged phospholipids is reversible
and can be reversed by ethylenediamine tetraacetic acid (EDTA). The
proteins according to the invention are capable of displacing
factor X.sub.a and prothrombin from a negatively charged
phospholipid surface.
The invention relates particularly to anti-coagulant proteins which
do not inactivate the coagulation factors and have molecular
weights of approximately 70.times.10.sup.3, 60.times.10.sup.3,
34.times.10.sup.3, or 32.times.10.sup.3, of which the proteins with
a molecular weight of 34.times.10.sup.3 or 32.times.10.sup.3 have a
single polypeptide chain.
The invention preferably relates to a family of anti-coagulant
proteins which do not inactivate the coagulation factors and are
characterized in that:
they are isolated from the walls of blood vessels in mammals and
are then substantially purified,
they are not glycoproteins,
they are not phospholipases,
they have an isoelectric point of pH 4.4-4.6,
the activity of the anti-coagulant proteins at 56.degree. C. is
thermally unstable,
the activity of the anti-coagulating proteins in citrated plasma
remains stable for some hours at 37.degree. C.,
the activity of the anti-coagulant proteins is not completely
destroyed by trypsin and/or chymotrypsin,
the activity of the anti-coagulant proteins is not affected by
collagenase and/or elastase,
they bind, via the divalent cations Ca.sup.2+ and Mn.sup.2+, to
negatively charged phospholipids which can be found in vesicles,
liposomes or etherosomes,
they bind via the divalent cations Ca.sup.2+ and Mn.sup.2+ to
negatively charged phospholipids which are coupled to
Spherocil,
the binding of the proteins to the negatively charged phospholipids
is reversible and can be removed by ethylenediamine tetracetic acid
(EDTA),
they displace factor X.sub.a and prothrombin from a negatively
charged phospholipid surface,
they inhibit the modified thrombin-time experiment,
they inhibit the modified, activated, partial thromboplastin-time
experiment,
they inhibit the non-modified prothrombin-time experiment,
they inhibit prothrombin activation by the coagulation factor
X.sub.a in the presence of negatively charged phospholipids and
Ca.sup.2+ in vitro,
they do not inhibit the biological and amidolytic activity of
factors X.sub.a and II.sub.a,
they inhibit the intrinsic X-activation by the factor IX.sub.a in
the presence of negatively charged phospholipids and Ca.sup.2+ in
vitro,
they inhibit the prothrombin activation of isolated, stimulated
blood platelets in vitro,
they inhibit the coagulation induced by the walls of the blood
vessels in vitro, and
the inhibition of prothrombin activation by factor X.sub.a induced
by the proteins is dependent on the concentration of phospholipids
and is reduced at high phospholipid concentrations.
In particular, the invention relates to VAC proteins substantially
free of any animal tissue, especially in substantially pure
form.
Suitable starting materials for the isolation of the VAC proteins
are the blood vessel walls and highly vascularized tissue of
various mammals, for example, cattle, rats, horses, and humans, as
well as endothelial cell cultures of these mammals. The arterial
walls of cattle, rats, horses, and humans and human umbilical veins
and arteries are particularly suitable.
The invention also relates to a process for preparing the proteins
of the invention using isolation and purification techniques. In a
procedure which is particularly suitable, the starting material is
homogenized and subjected to differential centrifugation. The
supernatant liquid obtained can then be further treated as follows
in any desired sequence. Undesirable contaminants can be
precipitated with ammonium sulfate. The supernatant is then further
purified by affinity chromatography, for example, using
hydroxyapatite; ion exchange chromatography, for example, using
DEAE-Sephacel; chromatograpy over a molecular sieve, such as
Sephadex G-100, and immunoabsorption chromatography, for example,
with polyclonal or monoclonal antibodies. Depending on the quality
of the starting material the purification process can be modified
or other purification procedures can be used such as, for example,
phospholipid vesicles.
In addition to the classic anti-thrombosis treatment, namely,
coagulants taken orally, more recently biosynthetic
tissue-plasminogen activator has been administered by the
intrasvascular route for cases of manifest thrombosis (N. Engl. J.
Med., 310: 609-513 (1984)).
The proteins according to the present invention are especially
suitable for preventing thrombosis, for example, during operations,
because of their blood coagulation-inhibiting properties while at
the same time inhibiting the coagulation-dependent aggregation of
platelets.
The present invention therefore also relates to the use of the
proteins according to the invention as antithrombotic agents.
The invention further relates to pharmaceutical compositions which
comprise at least one protein according to the invention in
association with a pharmaceutically acceptable carrier and/or
excipient.
The anti-coagulant proteins of the invention can be administered
parenterally by injection or by gradual perfusion over time. They
can be administered intravenously, intraperitoneally,
intrasmuscularly, or subcutaneously.
Preparations for parenteral administration include sterile or
aqueous or non-aqueous solutions, suspensions, and emulsions.
Examples of non-aqueous solvents are propylene glycol, polyethylene
glycol, vegetable oils such as olive oil, and injectable organic
esters such as ethyl oleate.
Aqueous carriers include water, alcoholic/aqueous solutions,
emulsions or suspensions, including saline and buffered media.
Parenteral vehicles include sodium chloride solution, Ringer's
dextrose, dextrose and sodium chloride, lactated Ringer's or fixed
oils. Intravenous vehicles include fluid and nutrient replenishers,
electrolyte replenishers, such as those based on Ringer's dextrose,
and the like. Preservatives and other additives can also be
present, such as, for example, antimicrobials, antioxidants,
chelating agents, inert gases, and the like. See, generally,
Remington's Pharmaceutical Science, 16th Ed., Mack, eds. 1980.
The invention also relates to a method for preparing a medicament
or pharmaceutical composition comprising the components of the
invention, the medicament being used for anti-coagulant
therapy.
Results from the isolation and purification of VAC from bovine
arteries are shown in Table A. Determination of the level of VAC
activity in the supernatant of the 100,000.times.g centrifugation
was erroneous owing to the presence of procoagulant activity. The
components responsible for this activity were found to be
precipitated with ammonium sulfate at a saturation level of 35%. It
was discovered that the supernatant solution obtained after
precipitation with 35% ammonium sulfate contained 100% VAC
activity. In order to precipitate this activity, the solution was
mixed with ammonium sulfate until 90% saturation was achieved. The
resulting precipitate containing the VAC proteins was bound to a
hydroxyapatite column in the presence of TBS (100 mM NaCl, 50 mM
Tris/HCl, pH 7.5). After washing, the VAC proteins were eluted from
this column with an increasing phosphate gradient. At low ion
concentration, the VAC proteins were bound to the DEAE-Sephacel
column. Elution of the VAC proteins from this column was done using
an increasing NaCl concentration gradient. In the final
purification step, the proteins were separated on the basis of
their molecular weight by gel filtration on Sephadex G-100. A
high-salt buffer was used as the eluant to minimize the interaction
of VAC with the Sephadex material. VAC was eluted from this column
in a volume of about 1.6 times the void volume of the column (see
FIG. 1). The total yield of VAC after this final purification was
35%. By SDS-PAGE, all G-100 fractions which showed VAC activity
were found to contain two polypeptides (molecular weight 34,000 and
32,000, respectively). In some cases, an additional fraction with a
molecular weight of 60,000 showed VAC-activity.
Using SDS-PAGE, only peak fractions 138-140 were homogeneous in
relation to the two polypeptides. These fractions were used for all
other experiments concerning investigation of bovine VAC described
in the specification, with the exception of the experiments for
characterizing the binding of bovine VAC to phospholipid
liposomes.
In G-100 fraction 139, 3.4% of the VAC activity was found to have a
specific activity of 1480 units per mg of protein by means of a
one-stage coagulation test (see Example 1 and Table A). This
fraction contained no detectable quantity of phospholipid. An
extinction coefficient of ##EQU1## was calculated for this purified
VAC preparation from the absorption at 280 nm and from the protein
content.
As shown in FIG. 2, the two polypeptides with molecular weights of
34,000 and 32,000, which are present in the purified protein
material from bovine arteries and to which VAC activity has been
ascribed, have a single polypeptide chain. Using Schiff's reagent
with basic fuchsin, it was established that both proteins contain
few carbohydrate groups. Moreover, no gamma-carboxyglutamate (Gla)
residues could be found in either protein. Isoelectric focusing
(Example 1) showed that both proteins migrate in a single band
corresponding to an isoelectric point of 4.4 to 4.6 (FIG. 3).
The VAC activity was obtained from the PAG plate by elution of this
band from the gel. Analysis of the eluant with SDS-PAGE again
showed the presence of the two proteins. It was thus possible to
confirm that both proteins migrate in a single band in the pH
gradient of the PAG plate. In order to check the method, human
hemoglobin (Hb) was also investigated by isoelectric focusing. The
value of 6.8 found for Hb agrees with the value given in the
literature (see FIG. 3).
Binding experiments showed that the VAC activity can bind to
negatively charged phospholipid membranes. This binding takes place
in the presence of Ca.sup.2+ and Mn.sup.2+, but not in the presence
of Mg.sup.2+ or in the absence of divalent metal ions (see Table
B). This binding of VAC activity to liposomes is reversible using
EDTA.
Using SDS-PAGE, it was possible to show that both proteins can bind
to liposomes in the presence of Ca.sup.2+ and that this binding is
disrupted when EDTA is added (see FIG. 4). This is yet another
indication that VAC activity can be ascribed to these two
proteins.
On storage in tris-buffered saline (TBS) containing 10% glycerol,
VAC activity is stable at -70.degree. C. for at least three months,
at 0.degree. C. for at least 12 hours, and at 37.degree. C. for at
least half an hour. At 56.degree. C., the activity disappears
within two minutes.
The activity of VAC prolongs the coagulation time in a one-stage
coagulation experiment (Example 1) in which coagulation is
triggered with thromboplastin from bovine brains (BTP). Replacement
of BTP in this experiment with purified bovine thrombin or purified
bovine factor X.sub.a showed that VAC prolongs the coagulation time
only if factor X.sub.a is used to initiate coagulation; coagulation
induced by thrombin is not affected by VAC. This indicates that VAC
directly inhibits the factor X.sub.a activity or that there is some
interaction with the prothrombinase complex.
In further testing, an amidolytic thrombin formation test using
purified bovine factor X.sub.a and prothrombin was carried out.
FIG. 5 shows that when prothrombin is activated in the presence of
Ca.sup.2+ and phospholipid by means of factor X.sub.a to form
thrombin, VAC inhibits the prothrombin activation and the degree of
inhibition is dependent on the concentration of VAC. Moreover, the
inhibiting effect of VAC is greater at a lower concentration of
phospholipid.
FIG. 6 shows the phospholipid dependency of the VAC-induced
inhibition of prothrombin activation. It is significant that at a
phospholipid concentration of zero the prothrombin activation by
factor X.sub.a is not inhibited by VAC. Control tests showed that
VAC itself has no effect on the system of measurement.
Incubation of 5 uM phospholipid
[1,2-dioleoylsn-glycero-3-phosphoserine
(PS)/1,2-dioleoyl-sn-glycerol-3-phosphocholine (PC), 1:4 mol/mol]
with 107 ug/ml VAC (specific activity: 1,300 units per mg) and 10
mM Ca.sup.2+ reduced the procoagulant activity within 3 minutes at
37.degree. C. This shows that VAC has no phospholipase
activity.
In contrast to antithrombin III (AT-III), VAC has no effect on the
amidolytic activity of purified thrombin and no lasting effect on
factor X.sub.a activity, as measured with the chromogenic substrate
S 2337
(N-benzoyl-L-isoleucyl-L-glutamyl-L-pipecolyl-glycyl-L-arginine-p-nitroani
lide-dihydrochloride) or S 2238
(H-D-phenylalanyl-L-pipecolyl-L-arginine-p-nitroanilide-dihydrochloride)
[see Table C]. This table also shows that the inactivation of
factor X.sub.a and thrombin by AT-III is not intensified by VAC.
Heparin, on the other hand, decisively increases inactivation of
thrombin and factor X.sub.a in the presence of AT-III. This shows
that VAC has neither a heparin-like activity nor at AT-III-like
activity.
The isolation of the anti-coagulant of the invention from human
tissue may be achieved by the same isolation procedure using, for
example, a homogenate of human umbilical cord arteries. In such an
homogenate, an anti-coagulant according to the present invention
has been discovered by its ability to prolong the clotting time in
a prothrombin time test. The anti-coagulation activity became
measurable after Sephadex G-100 fractionation of the arterial
homogenate [See Example 4]. From further isolation procedures, this
activity is associated with a water-soluble substance(s), that
carries an overall negative charge at pH 7.9.
Analysis of Sephadex G-75 fractions with gel electrophoresis has
shown a positive correlation between the intensity of the 32,000 MW
band and the prolongation of the clotting time as measured with a
modified prothrombin time test (MPTT) [See Example 4]. The
connection between the 32K-band and anti-coagulant activity is
demonstrated by the fact that only the 32K-band of the
polyacrylamide gel has anti-coagulant activity. In combination with
the findings that the anti-coagulant rapidly loses its activity
when incubated at 56.degree. C., and that proteolytic enzymes can
destroy its activity, it is likely that the anti-coagulant activity
is expressed by a single protein with a molecular weight of 32,000
daltons.
Trypsin, in contrast to protease type I, is a poor inactivator of
the anti-coagulant. This suggests that the anti-coagulant possesses
only a small number of lysine- and arginine-residues that are
accessible to trypsin. The nature of the anti-coagulant activity
has been studied by initiating coagulation in different ways.
Clotting, induced by either the vascular procoagulant, HTP (human
brain thromboplastin), or factor X.sub.a, is inhibited by the
anti-coagulant; thrombin-induced clotting, on the other hand, is
not. From these findings, one can conclude that the anti-coagulant
interferes with thrombin formation, not with thrombin action.
Prothrombinase reconstituted from purified factors and prothrombin
were used to further study the anti-coagulant mechanism [See
Example 4]. Under these experimental conditions, the anti-coagulant
can inhibit the activation of prothrombin by complete
prothrombinase (factor X.sub.a, factor V.sub.a, phospholipid,
Ca.sup.2+) and by phospholipid-bound factor X.sub.a (factor
X.sub.a, phospholipid, Ca.sup.2+) but not by free factor X.sub.a
(factor X.sub.a, Ca.sup.2+).
The time course for prothrombin activation in the presence of the
anti-coagulant indicate an instantaneous inhibition of prothrombin
activation which remains constant in time. This shows that the
anti-coagulant acts neither by a phospholipase, nor by a
proteolytic activity. The fact that the activation of prothrombin
by factor X.sub.a and Ca.sup.2+ is not affected by the
anti-coagulant at all, strongly indicates that the anti-coagulant
mechanism of the vascular compound differs from that of the well
known plasma protease inhibitors such as antithrombin III. Since
Walker et al., Biochim. Biophys. Acta, 571: 333-342 (1979), have
demonstrated that activated protein C does not inhibit prothrombin
activation by factor X.sub.a, Ca.sup.2+ and phospholipid, it can
also be concluded that this compound is not protein C and does not
belong in Group 2 described above.
Preliminary binding studies indicate that the vascular
anti-coagulant probably interferes with the lipid binding of factor
X.sub.a and/or prothrombin. Whether the ability of the
anti-coagulant to inhibit prothrombin activation completely
accounts for its prolongation of the prothrombin time remains to be
established.
The fact that this inhibitor can be found in various types of
arteries, but not in poorly vascularized tissue indicates that a
physiological modulator of hemostasis and thrombosis, active at the
vascular level, has been found.
On the absis of the properties and activities of VAC which have
been observed, the blood coagulation mechanism under the influence
of VAC may be interpreted.
VAC binds via Ca.sup.2+ ions to negatively charged phospholipids
which occur as a result of damage to the tissues and/or because of
the stimulation of blood platelets, and thereby reduces the binding
of specific coagulation factors (vitamin K-dependent coagulation
factors) to the negatively charged phospholipid surface which acts
as a catalytic surface for these coagulation factors (Biochem.
Biophys. Acta, 515: 163-205 (1985)). As a result, the
phospholipid-dependent blood coagulation reactions are inhibited by
VAC. On the basis of its mechanism of activity, VAC can be
categorized in Group 3 described above.
However, a critical difference between VAC and the other known
proteins of this group is that VAC does not hydrolyze phospholipids
and therefore does not decompose any essential membrane
structures.
Among the properties of VAC which have not hitherto been described
for any of the known anti-coagulants is the fact that the
anti-coagulation effect of VAC is dependent on the concentration of
phospholipids in the coagluation process. This dependency means
that the coagulation process which has been initiated, for example,
by slight damage to the wall of the blood vessel and/or by slight
activation of blood platelets, that is, by a thrombotic process,
can be inhibited by VAC. On the other hand, the coagulation process
which is triggered by severe damage to walls of blood vessels,
wherein phospholipids are present in high concentrations, is not
inhibitied by VAC, because of high phospholipid concentrations. The
danger of severe bleeding when using VAC is therefore extremely
small. This property of VAC is in contrast to all the previously
known anti-coagulants which render one or more of the coagulating
factors ineffective and thereby increase the risk of severe
bleeding.
Another surprising property of VAC is that it does not deactivate
the coagulating factors themselves. Consequently, the coagulating
factors can still perform their other functions. For example, some
active coagulating factors also play a non-hemostatic role in the
chemotaxis of the inflammatory cells which participate in the
repair of damaged blood vessel walls.
This invention further describes a novel class of anti-coagulant
proteins which do not inactivate the coagulation factors. The
Examples serving to illustrate the invention and the properties
listed should not restrict the invention in any way. Anyone skilled
in the art will be able, without any inventive effort, to obtain
other proteins which have anti-coagulant properties without
inactivating the coagulation factors, using the method described.
These proteins also fall within the scope of protection of this
invention.
The abbreviations used in the invention have the following
meanings:
VAC: vascular anti-coagulant
PFP: platelet free plasma
TBS: 100 mM NaCl, 50 mM Tris/HCl, pH 7.5
EDTA: ethylenediamine tetraacetic acid
TBSE: TBS with 2 mM of EDTA
BTP: thromboplastin from bovine brains
HTP: thromboplastin from human brains
TBSA: TBS with 0.5 mg/ml of human serum albumin, pH 7.9
S 2337:
N-benzoyl-L-isoleucyl-L-glutamyl-L-pipecolyl-glycyl-L-arginine-p-nitroanil
ide-dihydrochloride
S 2238:
H-D-phenylalanyl-L-pipecolyl-L-arginine-p-nitroanilide-dihydrochloride
AT-III: human antithrombin III
S.A.: specific activity
Ole.sub.2 Gro-P-Cho: 1,2,-dioleolyl-sn-glycero-3-phosphocholine
Ole.sub.2 Gro-P-Ser: 1,2,-dioleolyl-sn-glycero-3-phosphoserine
The nomenclature of the blood coagulation factors used was that
recommended by the Task Force on Nomenclature of Blood Clotting
Zymogens and Zymogen Intermediates.
Having now generally described this invention, the same will be
better understood by reference to certain specific examples which
are included herein for purposes of illustration only and are not
intended to be limiting of the invention unless otherwise
specified. Particularly, it is noted that, in principle, the
present invention applies to all anti-coagulants from human and
other animal sources, provided that they satisfy the purity and
reactivity criteria, and also to preparations of the
above-described compounds obtained by methods other than those
disclosed herein.
EXAMPLE 1
Characterization of VAC
(a) Isolation and Purification of VAC
The chemicals for analytical SDS-PAGE and hydroxyapatite (HTP) were
obtained from Bio-Rad. Sephadex G-100 and G-75, DEAE-Sephacel and
the "Low Molecular Weight Calibration Kit" were obtained from
Pharmacia. The chromogenic substrates S 2337 and S 2238 were
obtained from Kabi Vitrum and the Diaflo PM-10 ultrafiltration
membrane was obtained from Amicon.
Bovine aortas were taken within half an hour after slaughtering the
animals. Bovine blood was collected in trisodium citrate (final
concentration 0.38% by weight) and centrifuged for 10 minutes at
ambient temperature at 2,000.times.g. The plasma containing few
blood platelets was then centrifuged again (15 minutes at
10,000.times.g). In this way, platelet-free plasma was obtained
(PFP).
The aortas from the animals were thoroughly rinsed with TBS (100 mM
NaCl, 50 mM Tris/HCl, pH 7.5) immediately after being removed. The
inner lining of the aortas was removed and homogenized using a
high-speed homogenizer, e.g., the Braun MX 32, in TBSE (TBS with 2
mM EDTA) containing soyabean trypsin inhibitor (16 mg/l) and
benzamidine (1.57 g/l).
The material homogenized from eight aortas and containing 20%
solids (weight/volume) was centrifuged for 60 minutes at
100,000.times.g. The supernatant was saturated with solid ammonium
sulfate to 30% saturation, stirred from 30 minutes, and then
centrifuged for 20 minutes at 12,000.times.g. The resulting
supernatant was saturated with solid ammonium sulfate to 90%
saturation, stirred for 30 minutes, and centrifuged for 20 minutes
at 12,000.times.g.
The precipitate was suspended in a small volume of TBS and dialyzed
with TBS containing benzamidine (1.57 g/l). The dialyzed fraction
was applied to a hydroxyapatite column (1.times.20 cm) which had
been equilibrated with TBS. The column was washed with four bed
volumes of TBS and the VAC proteins eluted with 200 ml of sodium
phosphate buffer (pH 7.5) using a linear gradient (0-500 mM). The
fractions containing VAC were combined and dialyzed against 50 mM
of NaCl with 20 mM of Tris/HCl at pH 7.5.
This same buffer was used to equilibrate a DEAE-Sephacel column
(3.times.5 cm) on which the dialyzed VAC material was
chromatographed. The column was washed with four bed volumes of the
equilibration buffer and the VAC eluted with 200 ml of NaCl
solution in 20 mM of Tris/HCl, pH 7.5, using a linear gradient
(50-300 mM). The fractions containing VAC were collected, dialyzed
with 500 mM NaCl in 20 mM of Tris/HCl at pH 7.5 and then
concentrated in an Amicon concentration cell using a PM-10
ultrafiltration membrane. The concentrate (2 ml) was applied to a
Sephadex G-100 column (3.times.80 cm) equilibrated with 500 mM NaCl
in 20 mM Tris/HCl, pH 7.5.
The eluate was collected in 2 ml fractions and the active fractions
dialyzed separately against TBS containing 10% by volume glycerol
and stored at -70.degree. C. The entire purification was carried
out at 0.degree.-4.degree. C.
b. Determining VAC Activity
Two different methods (see, generally, Harrison's Principles of
Internal Medicine, 10th Ed., Petersdorf et al., eds., 1983) were
used to determine the VAC activity:
(a) the one-stage coagulation test (modified prothrombin time
test)
(b) thrombin formation test.
The one-stage coagulation test was carried out as follows:
In a siliconized glass dish, 175 ul of the fraction to be tested,
or 175 ul of TBS as control, were stirred with 50 ul of PFP and 25
ul of dilute BTP (900 rpm). After incubation (3 minutes at
37.degree. C.), coagulation was initiated by adding 250 ul of
buffer which contained 80 mM NaCl, 20 mM CaCl.sub.2, and 10 mM
Tris/HCl, pH 7.5. Fibrin formation was recorded optically using a
"Payton Dual Aggregation Module" (Hornstra, G., Phil. Trans. R.
Soc. London B, 294: 355-371 (1981)). The coagulation time of the
control sample was 65 seconds. This test was used during
purification to examine the various fractions for the presence of
VAC activity. In order to determine the VAC yield during
purification, one unit of VAC activity was defined as the quantity
of VAC which prolongs the coagulation time in the above test to 100
seconds.
In some cases, BTP was replaced by purified bovine thrombin or the
purified bovine factor X.sub.a. In this semi-purified coagulation
system, the quantity of thrombin or factor X.sub.a used were such
that the coagulation time of the control sample was also 65
seconds.
The thrombin formation test was carried out as follows:
20 ul of purified bovine factor X.sub.a (150 nM), 30 ul of
CaCl.sub.2 (100 mM), 30 ul of dilute VAC and 30 ul of
PS/PC-phospholipid membrane (the final concentrations are given in
the legend accompanying FIG. 6) were placed in a plastic dish
containing 181 ul TBSA (TBS with 0.5 mg/ml human serum albumin, pH
7.9).
This mixture was stirred for 3 minutes at 37.degree. C. with a
Teflon stirrer. Thrombin formation was initiated by adding 9 ul of
purified bovine factor II (33.33 uM). At various times, 50 ul
samples of the reaction mixture were added to a plastic dish
containing 900 ul of TBSE and 50 ul of chromogenic substrate S 2238
(5 mM, 37.degree. C.). The concentration of thrombin in the
reaction mixture was calculated from the change in extinction at
405 nm (Kontron Spectrometer Uvikon 810), using a calibration curve
plotted from assays with known quantities of purified bovine
thrombin. The percent inhibition caused by VAC was defined as
follows: ##EQU2## wherein "a" is the rate of thrombin formation in
the absence of VAC in nM II.sub.a /min, and "b" is the rate of
thrombin formation in the absence of VAC in nM II.sub.a /min.
The vitamin K-dependent factors prothrombin and factor X.sub.a were
obtained by purification of citrated bovine plasma (cf. Stenflo,
J., J. Biol. Chem., 251: 355-363 (1976)). After barium citrate
absorption and elution, fractionation with ammonium sulfate, and
chromatography on DEAE-Sephadex, there were two protein fractions
which contained a mixture of prothrombin and factor IX or factor X.
Factor X was activated using the method of Fujikawa et al.,
Biochemistry, 11: 4882-4891 (1972) and using RVV-X (Fujikawa et
al., Biochemistry, 11: 4892-4899 (1972)). Prothrombin was separated
from factor IX by heparinagarose affinity chromatography (Fujikawa
et al., Biochemistry, 12: 4938-4945 (1973)). The
prothrombin-containing fractions from the heparin-agarose column
were combined and further purified using the method of Owens et
al., J. Biol. Chem., 249: 594-605 (1974). The concentrations of
prothrombin and factor X.sub.a were determined using the method of
Rosing et al., J. Biol. Chem., 255: 274-283 (1980). BTP was
prepared by the method of Van Dam-Mieres et al., Blood Coagulation
Enzymes, Methods of Enzymatic Analysis, Verlag Chemie GmbH,
Weinheim. The protein concentrations were determined according to
Lowry et al., J. Biol. Chem., 193: 265 (1951).
C. Preparation of Phospholipids, Phospholipid Membranes and
Phospholipid Liposomes
Phospholipids were prepared using
1,2-dioleoyl-sn-glycero-3-phosphocholine (18:1.sub.cis
/18:1.sub.cis -PC) and 1,2-dioleoyl-sn-glycero-3-phosphoserine
(18:1.sub.cis /18:1.sub.cis -PS), as described by Rosing et al., J.
Biol. Chem., 255: 274-283 (1980). Separate phospholipid membranes
of PC and PS consisting of two layers were prepared using
ultrasound as described by Rosing et al., J. Biol. Chem., 255:
274-283 (1980). A supply of phospholipid liposomes was prepared by
dissolving the appropriate amount of phospholipid in chloroform
which was evaporated using nitrogen. The residual phospholipid was
suspended in TBS containg 5% glycerol, carefully mixed with a few
glass beads for 3 minutes, then centrifuged for 10 minutes at
10,000 xg. The above solution was discarded and the residue
carefully resuspended in TBS containing 5% glycerol. In this
manner, the phospholipid-liposome supply solution was obtained.
These liposomes were stored at ambient temperature. The
phospholipid concentration was determined by phosphate analysis
according to Bottcher et al., Anal. Chim. Acta., 24: 203-207
(1961).
Gel electrophoresis on plates in the presence of SDS was carried
out according to the method described by Laemmli, Nature, 227:
680-685 (1970) using a gel which contained 10% by weight
acrylamide, 0.27% by weight N,N.sup.3 -methylene-bisacrylamide and
0.1% by weight SDS. In gel samples with reduced disulfide bridges,
5% by weight beta-mercapto-ethanol was present. The gels were
stained as follows:
(1) 0.25% by weight Coomassie Blue R-250 in 50% by weight ethanol
and 15% by weight acetic acid, and decolorized with 10% by weight
ethanol and 10% by weight acetic acid, or
(2) with Schiff's reagent prepared from basic fuchsin (Merck) by
the method of Segrest et al., described in Methods in Enzymology,
Vol. 28, 54-63 (1972), or
(3) with silver as described by Merril et al. in Electrophoresis,
3: 17-23 (1982).
The isoelectric pH measurements of proteins were done using thin
layer polyacrylamide gels which contain ampholine carrier ampholyte
(PAG plates, LKB) at a pH range of 3.5-9.5 in accordance with the
manufacturer's instructions. The pH gradient in the gel was
determined immediately after electrofocusing by cutting off a strip
of the gel along a line between the anode and the cathode. The
electrolytes were eluted from each strip using distilled water and
the pH measured with a combined glass electrode.
The Gla determination was carried out by HPLC on a "Nucleosil 5SB"
column (CHROMPACK) using the method of Kuwada et al., Anal.
Biochem., 131: 173-179 (1983).
EXAMPLE 2
Coupling of Phospholipids to Spherocil
The required phospholipids were dissolved in chloroform and added
to the column material (Spherocil, Messrs. Rhone-Poulenc) at a
ratio of 5 mg of phospholipid per gram of Spherocil. The chloroform
was evaporated with N.sub.2 gas and the dry Spherocil phospholipid
was then washed with the buffer in which VAC had been suspended.
VAC binds to Spherocil-coupled phospholipid in the presence of
Ca.sup.++ and/or Mn.sup.++ when some of the phospholipids are
negatively charged.
EXAMPLE 3
50 ul of citrated/platelet-free plasma were mixed with 200 ul of
buffer (25 mM Tris/HCl, pH 7.5, 100 mM NaCl), containing kaolin
(catalyzes coagulation), inositin (phospholipid source) and VAC
were present. This mixture was incubated (3 minutes at 37.degree.
C.) and 250 ul of Ca.sup.++ buffer (200 mM Tris/HCl, pH 7.5, 80 mM
NaCl, 20 mM CaCl.sub.2) was added. The coagulation time was
measured as described in Example 1.
EXAMPLE 4
Isolation and Characterization of Anti-Coagulant From Human
Tissue
Human blood was collected by venipuncture in trisodium citrate (13
mM) and centrifuged at 2,000 xg for 10 minutes at room temperature.
The resulting plasma was recentrifuged at 1,000 xg for 15 minutes
in order to obtain platelet free plasma (PFP). A standard pool of
PFP was prepared by mixing plasma from several healthy donors.
Human umbilical cords were obtained within 15 minutes after
delivery. The arteries were immediately perfused with ice-cold
TBS-buffer, subsequently prepared free from the Jelly of Warton,
and homogenized in TBS using a whirl mixer (Braun MX32). A 10%
homogenate (w/v) was then fractionated.
Fractionation of the supernatant from a 10,000 xg centrifugation of
the homogenate on Sephadex G-100 results in a reproducible profile
(see FIG. 7). The fractions affecting the coagulation system as
measured with the MPTT are indicated in FIG. 7. Procoagulant
activity eluted with the void volume. This activity can only be
detected in the presence of factor VII in the MPTT, as indicated by
experiments in which human congenital factor VII-deficient plasma
was used. This establishes that this procoagulant is tissue
thromboplastin.
Certain fractions showed a distinct anti-coagulant activity. These
fractions were pooled and further purified with DEAE-Sephacel
chromatography (see FIG. 8A). The anti-coagulant bound to the
DEAE-Sephacel with 50 mM NaCl in 50 mM Tris/HCl, pH 7.9. Elution of
activity with a linear gradient of NaCl at pH 7.9 was achieved at
150-160 mM NaCl. The DEAE-fractions expressing anti-coagulant
activity were pooled and filtered using Sephadex G-75 (FIG. 8B).
The column (1.5.times.50 cm) was equilibrated with TBS and activity
was present in those fractions which corresponded to molecular
weights of about 30,000-60,000 daltons.
The MPTT was used as a quantitative assay for the determination of
the amount of anti-coagulant activity (see FIG. 9). One unit of
anti-coagulant activity was defined as that quantity which prolongs
the clotting time in the MPTT, with HTP (final concentration 95 ug
protein/ml) as initiator of coagulation, from its control value of
65 s to 100 s. With this assay, it was calculated that from 10 g
wet arterial tissue 2 mg protein with approximately 1,200 units
anti-coagulant activity can be isolated.
The modified prothrombin time test (MPTT) was carried out as
follows:
In a siliconized glass cuvette, 50 ul PFP was stirred at 37.degree.
C. with 150 ul TBS, 25 ul of a standard HTP-dilution, and 25 ul TBS
(control) or 25 ul of a fraction of the arterial homogenate. After
incubation for 3 minutes, coagulation was started at time zero with
the addition of 250 ul Ca.sup.2+ -buffer (80 mM NaCl, 20 mM
CaCl.sub.2 and 10 mM Tris/HCl, pH 7.9). Fibrin formation was
monitored optically (Payton Dual Aggregation Module). When factor
X.sub.a was utilized to initiate coagulation in the MPTT, HTP was
omitted and 25 ul purified factor X.sub.a was added together with
the 250 ul Ca.sup.2+ -buffer to the diluted PFP.
The modified thrombin time test (MTT) was carried out similar to
the X.sub.a -initiated MPTT described above, with the exception
that the X.sub.a -preparation was replaced by 25 ul of purified
thrombin.
Protease type I and trypsin (EC 3.4.2.1.4) were obtained from
Sigma. HTP was prepared from human brain as described by van Dam
Mieras et al., Methods of Enzymatic Analysis, 5: 352-365 (1984).
Factor X.sub.a, prothrombin and thrombin were purified from
citrated bovine blood as described by Rosing et al., J. Biol.
Chem., 255: 274-283 (1980). Factor V was purified from bovine blood
as described by Lindhout et al., Biochemistry, 21: 4594-5502
(1982). Factor V.sub.a was obtained by incubating factor V with
thrombin. Prothrombin concentrations were calculated from MW=72,000
and A.sub.280.sup.1% =9.6 (Owen et al., J. Biol. Chem. 249: 594-605
(1974), and factor V concentration was calculated from MW=330,000
and A.sub.280.sup.1% =9.6 (Nesheim et al., J. Biol. Chem., 254:
508-517 (1979). Factor X.sub.a and thrombin concentrations were
determined by active site titration (Rosing et al., J. Biol. Chem.,
253: 274-283 (1980). Other protein concentrations were determined
as described by Lowry et al., J. Biol. Chem., 193: 265 (1951).
Phospholipid and phospholipid vesicles were prepared using
Ole.sub.2 Gro-P-Cho(1,2-dioleoyl-sn-glycero-3-phosphocholine) and
Ole.sub.2 Gro-P-Ser(1,2-dioleoyl-sn-glycero-3-phosphoserine) as
described in Rosing et al., supra (1980). Single bilayer vesicles
composed of Ole.sub.2 Gro-P-Ser/Ole.sub.2 Gro-P-Cho (molar ratio
20:80) were prepared by sonication. Phospholipid concentrations
were determined by phosphate analysis according to the method of
Bottcher et al., Anal. Chim. Acta, 24: 203-207 (1961).
The time course of prothrombin activation was examined at different
concentrations of anti-coagulant. Mixtures of (X.sub.a, Ca.sup.2+),
(X.sub.a, phospholipid, Ca.sup.2+) or (X.sub.a, V.sub.a,
phospholipid, Ca.sup.2+) were stirred with different amounts of the
anti-coagulant at 37.degree. C. in 50 mM Tris/HCl, 175 mM NaCl, 0.5
mg/ml human serum albumin at pH 7.9. After 3 minutes, prothrombin
activation was started by the addition of prothrombin. At different
time intervals, a 25 ul sample was transferred from the reaction
mixture into a cuvette (37.degree. C.), containing TBS, 2 mM EDTA
and 0.23 mM S 2238 (final volume: 1 ml). From the absorption change
at 405 nm (Kontron Spectrophotometer Uvikon 810), and a calibration
curve based on purified thrombin, the amount of thrombin formed was
calculated at different concentrations of anti-coagulant.
Phospholipid was added in the form of vesicles composed of
Ole.sub.2 Gro-P-Ser and Ole.sub.2 Gro-P-Cho with a molar ratio of
20:80.
Several fractions from G-75 chromatography were tested by MPTT and
analyzed using SDS-PAGE. The results (FIG. 10) showed that the
anti-coagulant has a molecular weight of approximately 32,000
daltons. The anti-coagulant activity of the 32K-band was confirmed
by slicing the polyacrylamide gel, eluting the protein and testing
the eluant for anti-coagulant activity as described above.
Anti-coagulant activity was found only in the eluant in the slice
corresponding to the 32K band. This activity was found to be stable
at 56.degree. C. and had a dose response relationship in the MPTT
similar to the starting material.
The G-75 fractions containing the highest anti-coagulant activity
were pooled and used for further characterization of the
anti-coagulant. Incubation of the anti-coagulant at 56.degree. C.
rapidly decreases its activity until after 2 minutes no activity
can be measured. The anti-coagulant loses its activity completely
within 2 hours upon incubation at 37.degree. C. with protease type
I, whereas trypsin has little effect on the anti-coagulant after an
incubation period of 3 hours (FIG. 11). The protease type I and the
trypsin concentration used in these experiments, completely
inactivate 2.5 nM thrombin in 15 minutes. The amounts of protease
type I and trypsin, carried over from the reaction mixtures to the
MPTT, have no effect on the control clotting time.
The MPTT is prolonged in the presence of the anti-coagulant (FIG.
12) both when initiated with HTP and when started with factor
X.sub.a. Thrombin-induced coagulation, however, is not
inhibited.
Because of these findings, we investigated the effect of the
anti-coagulant on the conversion of prothrombin to thrombin by
factor X.sub.a, factor V.sub.a, phospholipid and Ca.sup.2+. Under
the experimental conditions mentioned, thrombin formation is
inhibited by the anti-coagulant in a dose-dependent way (FIG. 13A).
The activation of prothrombin by factor X.sub.a, phospholipid and
Ca.sup.2+ in the absence of factor V.sub.a can be inhibited also by
the anti-coagulant (FIG. 13B). However, this inhibition is not
observed if the activation takes place in the absence of
phospholipid (FIG. 13C).
EXAMPLE 5
Polyclonal Antibodies Against VAC
Polyclonal antibodies against bovine VAC were raised in a rabbit.
Bovine VAC, purified according to the method as described in
Example 1, was mixed with equal amounts of complete Freund's
adjuvant. The mixture was injected subcutaneously into a rabbit.
After a period of 4 weeks, the rabbit was re-injected
subcutaneously with purified bovine VAC. The subcutaneous
injections were repeated twice at two-week intervals. Ten days
after the last injection, the rabbit was bled and the collected
blood was allowed to clot in order to obtain serum.
Immunoglobulins (Ig) were isolated from the serum according to the
following method:
(a) The serum was heated for 30 minutes at 56.degree. C.
(b) Subsequently, the serum was applied to DEAE-Sephacel, which was
equilibrated with 50 mM Tris, 100 mM NaCl, pH 8.2.
(c) The non-bound protein was precipiptated with (NH.sub.4).sub.2
SO.sub.4 at 50% saturation.
(d) The precipitated proteins were pelleted by centrifugation and
the pellet resuspended in 50 mM Tris, 100 mM NaCl, pH 7.9 and
dialyzed extensively against the same buffer.
(e) The resulting protein mixture contained anti-VAC Ig.
Following the procedure as described, protein fractions which
express VAC-activity were isolated from bovine aorta, bovine lung,
rat and horse aorta, and human umbilical cord arteries.
The proteins were separated by electrophoresis on a polyacrylamide
gel in the presence of dodecyl sulfate and under non-reduced
conditions. After completion of the electrophoresis, the proteins
were transferred from the gel to nitrocellulose sheets as described
by Towbin et al., Proc. Natl. Acad. Sci., USA, 76: 4350-4354
(1979). The sheets were incubated with the anti-VAC Ig and after
thorough washing the sheets were incubated with goat anti-rabbit Ig
coupled to horseradish peroxidase. The latter was visualized with
the peroxidase substrate diamine bezidine tetrahydrochloride.
A brown band on the nitrocellulose sheet, after completion of the
described procedure, indicated the presence of goat anti-rabbit Ig.
Furthermore, on this spot were present anti-VAC Ig and proteins to
which the anti-VAC Ig was bound.
Immunoblots of proteins with VAC activity, isolated from bovine
aorta, bovine lung, rat and horse aorta, and human umbilical cord
arteries are presented in FIG. 14.
These results show that by essentially using the isolation
procedure as described, a protein fraction with VAC activity can be
obtained from bovine aorta, bovine lung, rat and horse aorta, and
human umbilical cord arteries. Moreover, the isolated protein
fractions with VAC activity contain proteins, with MW of
approximately 32,000, 34,000, and 70,000, that react with anti-VAC
Ig raised against purified bovine VAC in rabbits.
EXAMPLE 6
Purification of VAC, Using Large Volume Phospholipid Vesicles
Large volume phospholipid vesicles (LVV), composed of
1,2-dioleoyl-sn-glycero-3-phosphoserine (PS) and
1,2-dioleoyl-sn-glycero-3-phosphocholine (PC), were prepared by the
method of P. van de Waart et al., Biochemistry, 22: 2427-2432
(1983).
For the purification step, LVV containing PS/PC (molar ratio 20:80)
was used. Other molar ratios can be used as long as negatively
charged phospholipids are present. The chain length of the fatty
acids in the phospholipids can also be varied.
LVV, .+-.1 mM phospholipids in 50 mM Tris/HCl, 100 mM NaCl, pH 7.9,
were mixed with an equal volume of a protein fraction containing
VAC activity. The proteins were in 50 mM Tris/NaCl, 10 mM
CaCl.sub.2, pH 7.9. The mixture was allowed to stand for 5 minutes
at ambient temperature. Subsequently, the mixture was centrifuged
for 30 minutes at 20,000.times.g. The pellet was resuspended in 50
mM Tris/HCl, 100 mM NaCl, 10 mM CaCl.sub.2, pH 7.9, and
recentrifuged. The resulting pellet was then resuspended in 50 mM
Tris/HCl, 10 mM ethylenediamine tetraacetic acid (EDTA), pH 7.9,
and recentrifuged. The resulting supernatant contained the VAC
activity.
The above described procedure is an efficient purification step in
the procedure to obtain purified VAC.
TABLE A ______________________________________ Summary of the
Purification of VAC from Inner Coat of Bovine Aorta Specific
Purification Protein.sup.a VAC.sup.b Activities Yield Degree of
Step mg Units units/mg % Purification
______________________________________ Supernatant 630 19.000 31.0
100 1.0 liquid with 35% (NH.sub.4).sub.2 SO.sub.4 Precipitate 470
19.000 40.4 97 1.3 with 90% (NH.sub.4).sub.2 SO.sub.4 Hydroxy- 206
17.300 84.0 89 2.7 apatite fraction DEAE 35.8 13.900 388 71 12.5
fraction Sephadex 0.45 0.666 1480 3.4 47.7 G-100 fraction 139
______________________________________ .sup.a Protein was
determined using the method of Lowry et al. (J. Biol. Chem., 193:
265 (1951). .sup.b The VAC units were determined using the onestage
coagulation test described in Example 1 by a series of test
dilutions. The coagulation tim of the control samples was 65
seconds. One unit of VAC activity was defined as the quantity of
VAC wh ich prolongs the coagulation time to 10 seconds.
TABLE B ______________________________________ Cation-Dependent
Binding of VAC to Negatively Charged Phospholipid Liposomes
t.sub.c, seconds.sup.a Supernatant Cation (10 mm) Liquid.sup.b
EDTA.sup.c ______________________________________ Control (no 180
.sup. N.D..sup.d liposomes) Control (no 174 64.8 cation) CaCl.sub.2
64.2 134 MgCl.sub.2 165 N.D. MnCl.sub.2 65.1 N.D.
______________________________________ .sup.a The coagulation time
(t.sub.c) was determined using the onestage coagulation test
described in Example 1. .sup.b 50 ul phospholipid liposomes (PS/PC;
50/50 mol/mp: 1 mm), 50 ul VA (250 ug/ml, specific activity = 700
units per mg), and 100 ul of TBS containing 5% glycerol and cation,
pH 7.5, were mixed at ambient temperature and centrifuged for 15 m
inutes at 15,000 xg. Supernatant liquid (25 ul) was diluted with
TBS to a final volume of 175 ul and teste using the onestage
coagulation test. The remainder of the supernatant liquid was
analyzed with SDSPAGE (FIG. 4). .sup.c The liposome precipitate was
suspended in 150 ul of TBS containing 5% glycerol and 10 mm EDTA,
pH 7.5. The suspension was centrifuged for 15 minutes at 15,000 xg.
The VAC activity of the supernatant was analyzed as described
above. .sup.d N.D. = not determined.
TABLE C ______________________________________ Effect of VAC on the
Amidolytic Activity of Factor X.sub.a and Factor II.sub.a VAC
(A.sub.405 /min .times. 10.sup.3).sup.a - +
______________________________________ X.sub.a 110.5 110.5 X.sub.a,
AT-III 80.0 81.5 X.sub.a, Heparin 110.5 109.0 X.sub.a, Heparin,
AT-III 47.5 .sup. N.D..sup.b II.sub.a 7.5 7.5 II.sub.a, AT-III 5.4
5.6 II.sub.a, Heparin 7.5 7.1 II.sub.a, Heparin, AT-III 0.56 N.D.
______________________________________ .sup.a The amidolytic
activity was measured as follows: Factor X.sub.a or Factor II.sub.a
was diluted with the abovementioned agents in TBSA. The reaction
mixture was stirred with a Tefloncoated stirrer in a plastic dis
(37.degree. C.). After 10 minutes, a sample of 100 ul (X.sub.a) or
50 ul (II.sub.a) was placed in another plastic dish (37.degree. C.)
which contained 800 ul of TBSE, 100 ul of TBSA, and 100 ul of S
2337 (2 mM) or 900 ul S 2238 (5 mM). The change in absorption at
405 nM was mea sured using a Kontron Spectrophotometer Uvikon 810
(37.degree. C.). The final concentrations of the various agents in
the reaction mixtures were as follows: Factor X.sub.a (18.7 nM);
Factor II.sub.a (1.5 nM); human ATIII (18.7 nM); heparin (1 unit
per ml); and VAC (10.7 ug/ml, specific activity: 1300 units/mg).
.sup.b N.D. = not determined
* * * * *